Fluorescence observation unit and fluorescence observation apparatus

ABSTRACT

A fluorescence observation apparatus includes a fluorescence observation unit, which includes a scanner that scans ultrashort pulsed laser light from a light source, a pupil projection lens that focuses the laser light scanned by the scanner, an image-forming lens that converts the focused laser light to substantially collimated light and causes the laser light to be incident on an objective lens, and a dichroic mirror that splits off fluorescence that is generated by the laser light focused on a sample by the objective lens and is collected by the objective lens. A photodetector detects the fluorescence split off by the dichroic mirror. A multi-mode optical fiber connects the fluorescence observation unit and the photodetector. A swiveling mechanism causes the fluorescence observation unit to swivel about an axis near the focal position of the objective lens. And a light-source optical fiber connects the light source and the fluorescence observation unit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 15/060,585 filed on Mar. 3, 2016, which is based on Japanese PatentApplication No. 2015-056294 filed on Mar. 19, 2015, the contents of bothof which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a fluorescence observation unit and afluorescence observation apparatus.

BACKGROUND ART

In the related art, there is a known observation apparatus for observinga living sample, such as a small laboratory animal, with whichobservation is performed by disposing an objective lens in variousdirections or at various positions with respect to the sample (forexample, see PTL 1).

In this observation apparatus disclosed in PTL 1, fluorescence generatedat a light-focusing position due to a multiphoton excitation effect byfocusing ultrashort pulsed laser light on a specimen is split off fromthe optical path of the ultrashort pulsed laser light immediately behindthe objective lens, is guided to an external photodetector by an opticalfiber, and is detected. By using a multiphoton-excitation-typeobservation apparatus like this, it is possible to focus the ultrashortpulsed laser light at a portion deep inside the sample, thus generatingfluorescence, whereby it is possible to acquire a fluorescence image ofthe portion deep inside the sample.

In the case of the multiphoton-excitation-type observation apparatus,the fluorescence collected by the objective lens is split offimmediately behind the objective lens, before returning to the scanner.Hence, when ultrashort pulsed laser light is scanned over the sample bythe scanner, the position of a fluorescence beam also variesaccordingly. Hence, in the case in which the scanning area scanned bythe scanner is large, it is difficult to make the split-off fluorescenceenter an optical fiber.

Furthermore, when the sample is a scattering material, such as abiological tissue, the fluorescence generated at the light-focusingposition of the ultrashort pulsed laser light is scattered by the samplein the course of returning to the objective lens. In order to obtain abright fluorescence image, it is necessary that fluorescence collectedfrom a wide area of the sample by using the objective lens is detected.

CITATION LIST Patent Literature

{PTL 1} Japanese Unexamined Patent Application, Publication No.2005-300655

SUMMARY OF INVENTION

A first aspect of the present invention is a fluorescence observationunit including: a scanner that scans ultrashort pulsed laser light froma light source; a pupil projection lens that focuses the ultrashortpulsed laser light scanned by the scanner; an image-forming lens thatconverts the ultrashort pulsed laser light, focused by the pupilprojection lens, to substantially collimated light and causes theultrashort pulsed laser light to be incident on an objective lens; and adichroic mirror that splits off, from the optical path of the ultrashortpulsed laser light, fluorescence that is generated at a light-focusingposition as a result of the ultrashort pulsed laser light being focusedon a sample by the objective lens and is collected by the objectivelens. The image-forming lens includes a first optical system havingpositive refractive power and disposed adjacent to the objective lens,and a second optical system having negative refractive power anddisposed at a position closer to the pupil projection lens than thefirst optical system is. The dichroic mirror is disposed between thefirst optical system and the second optical system.

A second aspect of the present invention is a fluorescence observationapparatus including: the fluorescence observation unit according to thefirst aspect; and a photodetector that detects the fluorescence splitoff by the dichroic mirror.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a schematic diagram of the overall configuration of afluorescence observation apparatus according to an embodiment of thepresent invention.

FIG. 2 illustrates a partial enlarged view of FIG. 1 for explainingdetection of fluorescence by a fluorescence observation unit provided inthe fluorescence observation apparatus in FIG. 1.

FIG. 3 illustrates a schematic diagram of the overall configuration of afirst modification of the fluorescence observation apparatus in FIG. 1for detecting multiple-wavelength fluorescence.

FIG. 4 illustrates a second modification of the fluorescence observationapparatus in FIG. 1, and illustrates a diagram for explaining the effectof an astigmatism correcting plate.

FIG. 5 illustrates a schematic diagram of the overall configuration of athird modification of the fluorescence observation apparatus in FIG. 1having a mechanism for observing the surface of a sample.

FIG. 6 illustrates a perspective view for explaining aninsertion/retraction operation of a dichroic mirror and a mirrorprovided in the fluorescence observation apparatus in FIG. 5.

DESCRIPTION OF EMBODIMENTS

A fluorescence observation unit 3 and a fluorescence observationapparatus 1 according to an embodiment of the present invention will bedescribed below with reference to the drawings.

As shown in FIG. 1, the fluorescence observation apparatus 1 accordingto this embodiment is a multiphoton-excitation laser scanning microscopeand includes a light source 2 that emits ultrashort pulsed laser lightin a near-infrared band; a fluorescence observation unit 3, according tothis embodiment, that irradiates a sample X with the ultrashort pulsedlaser light emitted from the light source 2 and collects fluorescencegenerated in the sample X; a photomultiplier tube (photodetector) 4 thatdetects the fluorescence collected by the fluorescence observation unit3; a first optical fiber (light-source optical fiber) 5 connecting thelight source 2 and the fluorescence observation unit 3; a second opticalfiber (optical fiber) 6 for guiding the fluorescence from thefluorescence observation unit 3 to the photomultiplier tube 4; a relayoptical system (relay lens) 7; a swiveling mechanism 25 that can swivelthe fluorescence observation unit 3; a controller 26 that inputsmovement instructions to the light source 2 and the fluorescenceobservation unit 3; and a monitor 27.

The fluorescence observation unit 3 includes a housing 8; and a scanner9, a pupil projection lens 10, an image-forming lens 11, a dichroicmirror 12, a mirror (optical-path deflecting member) 13, and a couplinglens 14, which are accommodated in the housing 8.

Furthermore, an objective lens 15 is replaceably mounted to thefluorescence observation unit 3.

The scanner 9 is, for example, a so-called proximity galvanometermirror, which includes two galvanometer mirrors 16 that are caused toswivel about non-parallel axes. The scanner 9 can two-dimensionally scanthe ultrashort pulsed laser light emitted from the light source 2 andguided by the first optical fiber 5.

The pupil projection lens 10 focuses the ultrashort pulsed laser lightscanned by the scanner 9 to form an intermediate image.

The image-forming lens 11 converts the ultrashort pulsed laser light,with which the pupil projection lens 10 has formed the intermediateimage, into substantially collimated light and causes the light to enterthe objective lens 15.

In this embodiment, the image-forming lens 11 includes a first opticalsystem 17 having positive refractive power and a second optical system18 having negative refractive power. The first optical system 17 isdisposed adjacent to the base end of the objective lens 15. The secondoptical system 18 is disposed at a position closer to the pupilprojection lens 10 than the first optical system 17 is, so as to bespaced from the first optical system 17 in the optical axis S direction.

In the drawings, although the first optical system 17 and the secondoptical system 18 are each illustrated as being formed of a single lens,the configurations of the optical systems 17 and 18 are not limitedthereto, and they may be each formed by combining a plurality of lensesso as to have positive refractive power or negative refractive power asa whole.

Specifically, the diverging ultrashort pulsed laser light entering theimage-forming lens 11 from the pupil projection lens 10 diverges furtheras it passes through the second optical system 18, is focused andconverted to substantially collimated light as it passes through thefirst optical system 17, and enters the objective lens 15.

The dichroic mirror 12 is formed in the shape of a parallel flat plateand is disposed at an angle of, for example, 45° with respect to theoptical axis S. The dichroic mirror 12 has such transmittancecharacteristics that it allows the ultrashort pulsed laser light to passtherethrough, while deflecting the fluorescence entering from theobjective lens 15 by substantially 90° to split the fluorescence offfrom the optical path of the ultrashort pulsed laser light.

The mirror 13 bends the optical path of the fluorescence such that thefluorescence split by the dichroic mirror 12 is further deflected bysubstantially 90° and is directed toward the opposite side of theobjective lens 15, along the direction parallel to the optical axis S ofthe objective lens 15.

The coupling lens 14 further focuses the fluorescence deflected by themirror 13 and makes the fluorescence enter an incident end of the secondoptical fiber 6.

The first optical fiber 5 is, for example, a single-mode fiber.

The second optical fiber 6 is a multi-mode fiber, and, in particular, ispreferably a liquid fiber. The core diameter, at the exit end, of thesecond optical fiber 6 is less than or equal to the effective diameterof the photomultiplier tube 4.

The relay optical system 7 temporarily converts the divergingfluorescence, propagated by the second optical fiber 6 and emitted fromthe exit end, to substantially collimated light, focuses thefluorescence again, and causes it to be incident on a photoelectricconversion surface 19 of the photomultiplier tube 4. The projectionmagnification of the relay optical system 7 from the exit end of thesecond optical fiber 6 to the photoelectric conversion surface 19 of thephotomultiplier tube 4 is set such that the diameter of the fluorescencebeam entering the photomultiplier tube 4 is within the effective area ofthe photoelectric conversion surface 19 of the photomultiplier tube 4and such that the incident NA to the photomultiplier tube 4 is within anallowable range of the photomultiplier tube 4. The preferred relaymagnification of the relay optical system 7 is, for example, 1×.

The swiveling mechanism 25 includes a slider (not shown) to which thehousing 8 of the fluorescence observation unit 3 is fixed, and acircular arc shaped rail 28 for guiding the slider. The rail 28 has acircular arc shape centered on the focal position of the objective lens15 a or the vicinity thereof. By moving the slider over the rail 28, thefluorescence observation unit 3 can be swiveled about the axis locatedat the focal position of the objective lens 15 or the vicinity thereof.

The controller 26 includes a computer (not shown) that executes acontrol program for the light source 2 and the scanner 9 of thefluorescence observation unit 3, a memory (not shown) storing thecontrol program executed by the computer, and an interface unit (notshown). The computer generates a two-dimensional image on the basis ofthe information about the position scanned by the scanner 9 according tothe executed control program and the brightness signal detected by thephotomultiplier tube 4. The memory stores the two-dimensional imagegenerated by the computer.

The interface unit includes a driver that transmits an operationinstruction from the computer to the scanner 9 and a signal processingcircuit that converts the signal detected by the photomultiplier tube 4from analog to digital and transmits the signal to the computer.

The monitor 27 displays the two-dimensional image generated by thecomputer.

The operation of the fluorescence observation unit 3 and fluorescenceobservation apparatus 1 according to this embodiment, configured asabove, will be described below.

When a fluorescence image of a relatively deep portion in the sample Xis to be acquired using the fluorescence observation apparatus 1according to this embodiment, ultrashort pulsed laser light is generatedby the light source 2, in response to the operation instruction from thecontroller 26. The ultrashort pulsed laser light generated by the lightsource 2 is guided to the fluorescence observation unit 3 via the firstoptical fiber 5.

The ultrashort pulsed laser light entering the fluorescence observationunit 3 through the first optical fiber 5 is two-dimensionally scanned bythe scanner 9 according to the operation instruction from the controller26 transmitted via the driver, is focused by the pupil projection lens10, is converted into substantially collimated light by theimage-forming lens 11, and then enters the objective lens 15. Theultrashort pulsed laser light focused at the focal position of theobjective lens 15 increases the photon density in a very limited areanear the focal position and excites a fluorescent substance existing inthat area to cause fluorescence to be generated.

The generated fluorescence is radiated in all directions, as shown inFIG. 2, and is scattered within the sample X. Of this scatteredfluorescence, the fluorescence radiated from the surface of the sample Xtoward the objective lens 15 is collected by the objective lens 15.

In this case, with the fluorescence observation unit 3 according to thisembodiment, because the image-forming lens 11 in which the first opticalsystem 17 having positive refractive power and the second optical system18 having negative refractive power are arranged at a distance from eachother in the optical axis S direction is employed, an advantage isobtained that it is possible to reduce the rear-side focal length and tomake the image-forming lens 11 itself have a compact configuration,unlike the case of the typical microscope, where a convex-concavedoublet lens is used as the image-forming lens 11.

Furthermore, because the fluorescence is split off by the dichroicmirror 12 that is disposed between the first optical system 17 and thesecond optical system 18, it is possible to split off the fluorescencethat is focused with a strong refractive power, compared with a casewhere the fluorescence passing through the whole image-forming lens 11is split off.

Specifically, the fluorescence generated at the focal position of theultrashort pulsed laser light spreads around the focal position due toscattering inside the surrounding sample X. Because the spreadfluorescence is also generated in the focal position of the ultrashortpulsed laser light, it is desirable to collect as much fluorescence aspossible.

As shown in FIG. 2, when the ultrashort pulsed laser light is focused ateach position in the scanning range of the ultrashort pulsed laserlight, scattered fluorescence enters the objective lens 15 from a widerarea including the scanning range. The light entering the objective lens15 from a wide area like this is collected by the objective lens 15 andthen diverges toward the image-forming lens 11. Because the firstoptical system 17 disposed in the vicinity of the objective lens 15 hashigher positive refractive power than a typical image-forming lens, thelight enters a narrow area of the dichroic mirror 12, in the form ofconverging light the central axis of which coincides with the opticalaxis S of the objective lens 15. Thus, even when the dichroic mirror 12is small, it is possible to split off the fluorescence coming from awide area of the sample X from the optical path of the ultrashort pulsedlaser light.

More specifically, it is possible to collect off-axis fluorescenceemitted from the objective lens 15 toward the image-forming lens 11 at alarge angle with respect to the optical axis S by deflecting, with thefirst optical system 17, the fluorescence in the direction parallel tothe optical axis S and to guide the fluorescence to the photomultipliertube 4.

As a result, with the fluorescence observation unit 3 and fluorescenceobservation apparatus 1 according to this embodiment, it is possible toefficiently collect the fluorescence generated at the focal position ofthe ultrashort pulsed laser light.

The fluorescence split off by the dichroic mirror 12 is deflected by themirror 13, is further focused by the coupling lens 14, and is made toenter the second optical fiber 6.

Because the second optical fiber 6 is made of a multi-mode fiber, inparticular, a liquid fiber, the core of the optical fiber 6 is large.Thus, it is possible to make the fluorescence focused by the couplinglens 14 efficiently enter the second optical fiber 6.

Specifically, with the fluorescence observation unit 3 according to thisembodiment that causes the fluorescence coming from a wider area beyondthe scanning range of the scanner 9 to be constantly incident on thesecond optical fiber 6, even when the ultrashort pulsed laser light isscanned by the scanner 9, the fluorescence entering the second opticalfiber 6 does not fluctuate and can be made to efficiently enter thesecond optical fiber 6.

The fluorescence guided by the second optical fiber 6 is emitted fromthe exit end of the second optical fiber 6, is relayed by the relayoptical system 7, and is incident on the photoelectric conversionsurface 19 of the photomultiplier tube 4, where it is detected as abrightness signal. The detected brightness signal is converted fromanalog to digital by the signal processing circuit and is transmitted tothe computer. The computer generates a two-dimensional image on thebasis of the transmitted brightness signal and the information about theposition scanned by the scanner 9, and the generated two-dimensionalimage is stored in the memory. As a result, it is possible to displaythe two-dimensional image stored in the memory on the monitor 27 asnecessary and to observe the fluorescence of the sample X.

Furthermore, the relay magnification of the relay optical system 7 isset such that the core diameter, at the exit end, of the second opticalfiber 6 is projected within the effective area of the photoelectricconversion surface 19. In this case, by setting the relay magnificationof the relay optical system 7 as viewed from the photomultiplier tube 4side to substantially lx or less, the inclination of the fluorescenceentering the photoelectric conversion surface 19 is reduced, which isadvantageous for preventing a decrease in intensity of detected lightdue to the angular dependence of the detection sensitivity.

Furthermore, because the core diameter, at the exit end, of the secondoptical fiber 6 is less than or equal to the effective diameter of thephotomultiplier tube 4, all of the fluorescence guided by the secondoptical fiber 6 is incident on the photoelectric conversion surface 19of the photomultiplier tube 4. Thus, it is possible to efficientlydetect the fluorescence, without being influenced by the entranceposition dependence.

Furthermore, in this embodiment, because the fluorescence deflected by90° by the dichroic mirror 12 is further deflected by 90° by the mirror13, it is possible to easily make the fluorescence, focused by thecoupling lens 14, enter the second optical fiber 6 fixed to the housing8 so as to extend in the direction substantially parallel to the opticalaxis S of the objective lens 15.

Furthermore, in this embodiment, even when the fluorescence observationunit 3 is swiveled by the swiveling mechanism 25 for causing thefluorescence observation unit 3 to swivel about the axis at the focalposition of the objective lens 15 or the vicinity thereof, the secondoptical fiber 6 does not inhibit such a movement, and it is possible tomake the space around the objective lens 15 more compact, ensuring alarge swiveling angle.

Although the case where single-wavelength fluorescence is acquired hasbeen described in this embodiment, when multi-wavelength fluorescence isacquired simultaneously, as shown in FIG. 3, dichroic mirrors(light-splitting dichroic mirrors) 20 may be disposed in the opticalpath of the collimated light formed by the relay optical system 7 tosplit the fluorescence according to the wavelengths and to detect thesplit-off fluorescences with separate photomultiplier tubes 4 a, 4 b,and 4 c.

In this embodiment, because the dichroic mirror 12, formed of a parallelflat plate, is disposed between the first optical system 17 and thesecond optical system 18, the ultrashort pulsed laser light passingthrough the dichroic mirror 12 is refracted as it passes through thedichroic mirror 12 and is shifted (astigmatism) in the direction inwhich the dichroic mirror 12 is inclined. To counter this problem, asshown in FIG. 4, an astigmatism correcting plate 21, which is formed ofa glass parallel flat plate having the same thickness as the dichroicmirror 12 and inclined by the same angle as the dichroic mirror 12 butin the opposite direction, may be provided. By doing so, it is possibleto prevent a shift in focal position of the ultrashort pulsed laserlight due to the dichroic mirror 12.

In this embodiment, as shown in FIG. 6, a mirror (observation-lightdeflecting member) 22 inclined in a direction different from thedirection in which the dichroic mirror 12 is inclined may be provided,and an insertion/retraction mechanism (not shown) that moves thedichroic mirror 12 and the mirror 22 in a direction as indicated by anarrow Y to alternatively dispose the mirrors 12 and 22 on the opticalaxis S of the objective lens 15 may be provided. Theinsertion/retraction mechanism is, for example, a linear motionmechanism. In this case, as shown in FIG. 5, the fluorescenceobservation unit 3 includes an image-acquisition element 23 thatcaptures an image of the light reflected by the mirror 22. In thefigure, reference sign 24 denotes a mirror.

The image-acquisition element 23 is connected to the controller 26. Thememory stores a camera-observation image acquired by theimage-acquisition element 23.

With this configuration, in a state in which the dichroic mirror 12 isdisposed on the optical axis S of the objective lens 15 by actuating theinsertion/retraction mechanism, it is possible to irradiate the sample Xwith the ultrashort pulsed laser light and to detect the fluorescence,as described above. On the other hand, in a state in which the mirror 22is disposed on the optical axis S of the objective lens 15 by actuatingthe insertion/retraction mechanism, by stopping the irradiation with theultrashort pulsed laser light and by irradiating the sample X with whitelight from outside the fluorescence observation unit 3, the reflectedlight from the sample X collected by the objective lens 15 is reflectedby the mirrors 22 and 24, and an image of the reflected light iscaptured by the image-acquisition element 23. In this way, it ispossible to acquire the camera-observation image of the surface of thesample X.

By acquiring the camera-observation image of the surface of the sampleX, by positioning the fluorescence observation unit 3 with respect tothe sample X, while confirming the observation position by displayingthe acquired camera-observation image on the monitor 27, and byirradiating the sample X with the ultrashort pulsed laser light from thelight source 2, with the dichroic mirror 12 being inserted by actuatingthe insertion/retraction mechanism, it is possible to acquire a brightfluorescence image.

Although the fluorescence split off by the dichroic mirror 12 is guidedto the photomultiplier tube 4 through the second optical fiber 6 in thisembodiment, instead, the photomultiplier tube 4 may be fixed to thehousing 8, and the fluorescence may be detected by the photomultipliertube 4, without passing through the optical fiber 6.

Similarly, although the reflected light from the surface of the sampleX, deflected by the mirror 22, is deflected by the mirror 24, instead,an image of the reflected light may be directly captured by theimage-acquisition element 23 after the reflected light is deflected bythe mirror 22.

The following aspects of the invention are derived from theabove-described embodiments and modifications thereof.

A first aspect of the present invention is a fluorescence observationunit including: a scanner that scans ultrashort pulsed laser light froma light source; a pupil projection lens that focuses the ultrashortpulsed laser light scanned by the scanner; an image-forming lens thatconverts the ultrashort pulsed laser light, focused by the pupilprojection lens, to substantially collimated light and causes theultrashort pulsed laser light to be incident on an objective lens; and adichroic mirror that splits off, from the optical path of the ultrashortpulsed laser light, fluorescence that is generated at a light-focusingposition as a result of the ultrashort pulsed laser light being focusedon a sample by the objective lens and is collected by the objectivelens. The image-forming lens includes a first optical system havingpositive refractive power and disposed adjacent to the objective lens,and a second optical system having negative refractive power anddisposed at a position closer to the pupil projection lens than thefirst optical system is. The dichroic mirror is disposed between thefirst optical system and the second optical system.

According to the first aspect of the present invention, the ultrashortpulsed laser light from the light source is scanned by the scanner, isfocused by the pupil projection lens, is converted into substantiallycollimated light by the image-forming lens, and is made incident on theobjective lens. The ultrashort pulsed laser light entering the objectivelens is focused on the sample disposed at the focal position of theobjective lens, and the photon density is increased at thislight-focusing position. As a result, a fluorescent substance existingin the sample is excited, and fluorescence is generated.

The fluorescence generated at the focal position of the objective lensscatters in all directions in the sample, and only the fluorescenceemitted from the surface of the sample in the direction in which thefluorescence enters the objective lens is collected by the objectivelens. Thereafter, the fluorescence is converted to converging light bythe first optical system, which has positive refractive power andconstitutes the image-forming lens, and fluorescence entering the firstoptical system from outside the optical axis is also split off from theoptical path of the ultrashort pulsed laser light by the dichroicmirror, in a state being refracted toward the optical axis direction.

Specifically, when the light-focusing position of the ultrashort pulsedlaser light on the sample is moved by the scanner, the fluorescence beamemitted from the focal position and collected by the objective lensfluctuates around the pupil position of the objective lens. However, byallowing the fluorescence to pass through the first optical systemhaving positive refractive power, it is possible to reduce the amount offluctuation.

Furthermore, the fluorescence entering the objective lens from arelatively wide area around the focal position is also converted toconverging light having a smaller diameter by passing through the firstoptical system having positive refractive power and is split off fromthe optical path of the ultrashort pulsed laser light by the dichroicmirror.

Hence, it is possible to split off, with the dichroic mirror, thefluorescence that cannot be split off in the form of a collimated lightbeam emitted from the objective lens and to efficiently detect thefluorescence. Thus, even when the ultrashort pulsed laser light isfocused at a deeper position in the sample, a bright and sharpfluorescence image can be acquired.

According to the first aspect of the present invention, the fluorescenceobservation unit may further include an astigmatism correcting platebetween the first optical system and the second optical system.

Because the fluorescence between the first optical system and the secondoptical system is converging light due to the first optical systemhaving positive refractive power, the light beam is shifted only in theinclination direction of the dichroic mirror due to the refractionoccurring when the ultrashort pulsed laser light passes through thedichroic mirror disposed so as to be inclined in one direction withrespect to the optical axis. By disposing an astigmatism correctingplate to shift the light beam in the direction opposite to the directionof the shift caused by the dichroic mirror, the shift of thelight-focusing position of the ultrashort pulsed laser can beeliminated.

A second aspect of the present invention is a fluorescence observationapparatus including: the fluorescence observation unit according to thefirst aspect; and a photodetector that detects the fluorescence splitoff by the dichroic mirror.

According to the second aspect of the present invention, by detecting,with the photodetector, the converging light formed by focusing thefluorescence fluctuating with a small amount of fluctuation andscattered in a wide area around the light-focusing position of theultrashort pulsed laser light on the sample, it is possible to acquire abright and sharp fluorescence image at a deeper position in the sample.

In the second aspect of the present invention, the fluorescenceobservation apparatus may further include an optical fiber that connectsthe fluorescence observation unit and the photodetector, and thefluorescence observation unit may include a coupling lens that focuses,at an end of the optical fiber, the fluorescence split off by thedichroic mirror.

With this configuration, by fixing the photodetector somewhere outsidethe fluorescence observation unit and by bending the optical fiber, itis possible to move the fluorescence observation unit. Hence, it ispossible to move the fluorescence observation unit while fixing thesample and to observe the various portions of the sample from variousdirections.

In the second aspect of the present invention, the fluorescenceobservation unit may include an optical-path deflecting member thatdeflects the fluorescence split off by the dichroic mirror in thedirection parallel to the optical axis of the objective lens and causesthe fluorescence to be incident on the coupling lens.

With this configuration, it is possible to deflect the optical path ofthe fluorescence, focused by the coupling lens, with the optical-pathdeflecting member and to cause the fluorescence to be incident on theoptical fiber disposed in the direction parallel to the optical axis ofthe objective lens. In other words, it is possible to extend the opticalfiber from the fluorescence observation unit in the direction parallelto the optical axis of the objective lens. As a result, even when thefluorescence observation unit is swiveled about the axis intersectingthe optical axis near the focal position of the objective lens, theoptical fiber does not inhibit such a movement, and it is possible tomake the space around the objective lens compact and to ensure a largeswiveling angle.

In the second aspect of the present invention, the fluorescenceobservation apparatus may further include a relay lens that relays, tothe photodetector, the fluorescence guided by the optical fiber andemitted from the other end of the optical fiber.

With this configuration, although the fluorescence entering one end ofthe optical fiber and guided by the optical fiber diverges at apredetermined divergence angle when emitted from the other end of theoptical fiber, the fluorescence is converted to substantially collimatedlight by the relay lens, is focused again, and is made incident on thephotodetector. The projection magnification of the relay lens from theother end of the optical fiber to a light-receiving surface of thephotodetector is set such that the diameter of the fluorescence beamentering the photodetector is within the effective area of thelight-receiving surface of the photodetector and such that the incidentnumerical aperture (incident NA) to the photodetector is within anallowable range of the photodetector. Thus, it is possible toefficiently detect the fluorescence, without being influenced by thedependence of the photodetector on the angle and the entrance position.

In the second aspect of the present invention, the relay lens mayconvert the fluorescence emitted from the other end of the optical fiberto a substantially collimated light beam, and the fluorescenceobservation apparatus may include a light-splitting dichroic mirror thatsplits the substantially collimated light beam, converted by the relaylens, according to the wavelength; and a plurality of the photodetectorsthat detect the fluorescences split by the light-splitting dichroicmirror, respectively.

With this configuration, the fluorescence is split according to thewavelength by the light-splitting dichroic mirror disposed in thecollimated light beam portion formed by the relay lens and is detectedby different photodetectors. Thus, it is possible to acquire a brightand sharp fluorescence image with each wavelength.

In the second aspect of the present invention, the fluorescenceobservation unit may include a housing in which the scanner, the pupilprojection lens, the image-forming lens, and the dichroic mirror aremounted, and the photodetector may be fixed to the housing.

With this configuration, it is possible to detect the fluorescence splitoff by the dichroic mirror with the photodetector directly or via a fewoptical elements, without using the optical fiber. Thus, it is possibleto reduce the loss of the fluorescence occurring every time thefluorescence passes through an optical element and to acquire a brighterfluorescence image.

In the second aspect of the present invention, the fluorescenceobservation unit may include an observation-light deflecting member thatdeflects the light from the sample, collected by the objective lens, ina direction different from the direction of the dichroic mirror; animage-acquisition element that acquires an image of the light deflectedby the observation-light deflecting member; and an insertion/retractionmechanism that alternatively disposes the dichroic mirror and theobservation-light deflecting member in the optical path of theultrashort pulsed laser light.

With this configuration, by actuating the insertion/retraction mechanismand disposing the dichroic mirror in the optical path of the ultrashortpulsed laser light between the first optical system and the secondoptical system, it is possible to split off, with the dichroic mirror,the fluorescence collected by the objective lens and further focused bythe first optical system from the optical path of the ultrashort pulsedlaser light and to detect the fluorescence with the photodetector. Onthe other hand, by actuating the insertion/retraction mechanism anddisposing the observation-light deflecting member in the optical path ofthe ultrashort pulsed laser light between the first optical system andthe second optical system, it is possible to deflect, with theobservation-light deflecting member, the light from the sample collectedby the objective lens and further focused by the first optical system inanother direction and to acquire an image of the light with theimage-acquisition element. Thus, for example, it is possible to acquirean image of the surface of the sample to confirm the observationposition and then switch to the dichroic mirror to observe thefluorescence image.

In the second aspect of the present invention, the fluorescenceobservation apparatus may further include a swiveling mechanism thatcauses the fluorescence observation unit to swivel about an axisintersecting the optical axis of the objective lens near the focalposition of the objective lens.

With this configuration, by actuating the swiveling mechanism, it ispossible to change the direction of the optical axis of the objectivelens while substantially fixing the focal position of the objective lenson the sample and to observe the sample from various directions, whilefixing the sample.

In the second aspect of the present invention, the optical fiber may bea multi-mode fiber.

In the second aspect of the present invention, the optical fiber may bea liquid fiber.

With this configuration, by using the multi-mode fiber having a largecore, in particular, by using the liquid fiber, it is possible to reducethe loss of the fluorescence fluctuating with a small amount offluctuation, the loss occurring when entering the optical fiber, and tocause the fluorescence to be efficiently incident on the photodetector.

In the second aspect of the present invention, the fluorescenceobservation apparatus may further include a light-source optical fiberthat connects the light source for generating the ultrashort pulsedlaser light and the fluorescence observation unit and guides theultrashort pulsed laser light emitted from the light source to thefluorescence observation unit.

With this configuration, by also fixing the light source for generatingthe ultrashort pulsed laser light somewhere outside the fluorescenceobservation unit and by bending the light-source optical fiber, it ispossible to easily move the fluorescence observation unit and to observethe sample from various positions, while fixing the sample.

REFERENCE SIGNS LIST

1 fluorescence observation apparatus

2 light source

3 fluorescence observation unit

4, 4 a, 4 b, and 4 c photomultiplier tube (photodetector)

5 first optical fiber (light-source optical fiber)

6 second optical fiber (optical fiber)

7 relay optical system (relay lens)

8 housing

9 scanner

10 pupil projection lens

11 image-forming lens

12 dichroic mirror

13, 24 mirror (optical-path deflecting member)

14 coupling lens

15 objective lens

17 first optical system

18 second optical system

20 light-splitting dichroic mirror

21 astigmatism correcting plate

22 mirror (observation-light deflecting member)

23 image-acquisition element

X sample

S optical axis

The invention claimed is:
 1. A fluorescence observation apparatuscomprising: (i) a fluorescence observation unit comprising: a scannerthat scans ultrashort pulsed laser light from a light source; a pupilprojection lens that focuses the ultrashort pulsed laser light scannedby the scanner; an image-forming lens that converts the ultrashortpulsed laser light, focused by the pupil projection lens, tosubstantially collimated light and causes the ultrashort pulsed laserlight to be incident on an objective lens; and a dichroic mirror thatsplits off, from an optical path of the ultrashort pulsed laser light,fluorescence that is generated at a light-focusing position as a resultof the ultrashort pulsed laser light being focused on a sample by theobjective lens and is collected by the objective lens; (ii) aphotodetector that detects the fluorescence split off by the dichroicmirror; (iii) an optical fiber that connects the fluorescenceobservation unit and the photodetector, wherein the optical fiber is amulti-mode fiber; (iv) a swiveling mechanism that causes thefluorescence observation unit to swivel about an axis intersecting anoptical axis of the objective lens near a focal position of theobjective lens; and (v) a light-source optical fiber that connects thelight source which generates the ultrashort pulsed laser light and thefluorescence observation unit and guides the ultrashort pulsed laserlight emitted from the light source to the fluorescence observationunit.
 2. The fluorescence observation apparatus according to claim 1,wherein the optical fiber that connects the fluorescence observationunit and the photodetector is a liquid fiber.
 3. The fluorescenceobservation apparatus according to claim 1, wherein the fluorescenceobservation unit includes an optical-path deflecting member thatdeflects the fluorescence split off by the dichroic mirror in adirection parallel to the optical axis of the objective lens.
 4. Thefluorescence observation apparatus according to claim 1, wherein thefluorescence observation unit includes a coupling lens that collects, atan end of the optical fiber, the fluorescence split off by the dichroicmirror.
 5. The fluorescence observation apparatus according to claim 2,further comprising a relay lens that relays, to the photodetector, thefluorescence guided by the optical fiber and emitted from an end of theoptical fiber.
 6. The fluorescence observation apparatus according toclaim 5, wherein the relay lens converts the fluorescence emitted fromthe end of the optical fiber to a substantially collimated light beam,and wherein the fluorescence observation apparatus further comprises: alight-splitting dichroic mirror that splits the substantially collimatedlight beam, converted by the relay lens, according to wavelength; and aplurality of the photodetectors, which detect fluorescences split by thelight-splitting dichroic mirror, respectively.
 7. The fluorescenceobservation apparatus according to claim 1, wherein the fluorescenceobservation unit further comprises: an observation-light deflectingmember that deflects light from the sample, collected by the objectivelens, in a direction different from a direction in which thefluorescence is directed by the dichroic mirror; an image-acquisitionelement that acquires an image of the light deflected by theobservation-light deflecting member; and an insertion/retractionmechanism that alternatively disposes the dichroic mirror and theobservation-light deflecting member in the optical path of theultrashort pulsed laser light.
 8. The fluorescence observation apparatusaccording to claim 1, wherein the dichroic mirror is disposed betweenthe objective lens and the scanner.
 9. The fluorescence observationapparatus according to claim 1, wherein the fluorescence observationunit further comprises a housing which houses the scanner, the pupilprojection lens, the image-forming lens, and the dichroic mirror. 10.The fluorescence observation apparatus according to claim 9, wherein theswiveling mechanism comprises a rail on which the housing is slidable.11. The fluorescence observation apparatus according to claim 10,wherein the rail has a circular-arc shape centered on the axisintersecting the optical axis of the objective lens near the focalposition of the objective lens.